鈣調素與Calneuron I對N型電壓依賴性鈣離子通道調控機制之研究

Abstract

N型電壓依賴性鈣離子通道（N-type Voltage gated Ca2+ channel, Cav2.2, N-type VGCC）最早發現於雞的背根神經節細胞（dorsal root ganglion）中，它會受到電位提升而活化開啟，而當鈣離子流入後，則會引發CDI（Ca2+ dependent inactivation）負回饋機制而抑制通道活性。過去對於CDI機制不太清楚，近年來已確定在L-及P/Q-type VGCCs中，是由鈣調素（Calmodulin）所調控的，但目前對於N-type VGCC的CDI機制還不瞭解。 為了了解N-type VGCC的CDI機制，我們將N-type VGCC與鈣調素同時表現於HEK293T中，紀錄鈣離子電流。實驗結果顯示，當細胞去極化達+20 mV時，在開啟後10 ms內會達到最大的鈣離子電流流入，之後在250 ms時，會下降至最大電流的13.3 ± 2.3%。如果將胞外溶液換為鋇離子，此不活化現象僅至82.4 ± 4.6%。而在鈣調素的N端以及C端EF hands突變株中，不活化現象則分別是43.1 ± 10.1% 及49.9 ± 8.2%。此外，如果我們將胞內溶液改為高緩衝液10 mM EDTA或BAPTA的情況下，也可恢復至87.2 ± 3.6% 及63.8 ± 8.2%。這些結果顯示，鈣調素的N端和C端皆可調控N-type VGCC的CDI，且此調控來自於廣泛性的鈣離子。另一方面，我們比較與鈣調素相似的鈣離子結合蛋白Calneuron I（Caln I）對N-type VGCC的調控。將Caln I與N-type VGCC同時表現於HEK293T中。實驗結果顯示，Caln I及其失去鈣離子結合功能的突變株在胞外鈣離子情況下都具有強烈的抑制作用，電流密度由-110.1 ± 29.9降至 -4.9 ± 5.0 及 -3.8 ± 2.3 pA/pF。然而，缺乏C端穿膜片段的突變株由於無法正常表現於細胞膜上，使其抑制作用降低至-33.1 ± 7.5 pA/pF。這些結果顯示N型電壓依賴性鈣離子通道可透過多種不同的鈣離子結合蛋白或機制調控其電生理活性。

N-type Voltage gated Ca2+ channel (Cav2.2, N-type VGCC) is first identified in chicken dorsal root ganglion neurons. VGCCs control the influx of Ca2+ into neurons in response to membrane depolarization. The channel could be inactivated by the Ca2+ influxed, a process called Ca2+ dependent inactivation (CDI). Calmodulin (CaM) has been identified to mediate the CDI of L- and P/Q-type VGCC. However, the mechanism of N-type VGCC CDI is not clear and studied in this report. CaM and N-type VGCC were co-expressed in HEK293T cells and the Ca2+ currents were studied in whole-cell configuration. When cell was depolarized to +20 mV, an inward current was evoked and reached a maxima in 10 ms. The current was then declined to 13.3 ± 2.3% of the peak current in 250 ms. Replacing the external Ca2+ with Ba2+, the inactivation was 82.4 ± 4.6%. When the N- or C-lobe EF hands of CaM was mutated and unable to chelate Ca2+, the inactivation was significantly changed to 43.1 ± 10.1 and 49.9 ± 8.2%, respectively. Increasing the buffer capacity of the internal solution with 10 mM EDTA or BAPTA, the inactivation was 87.2 ± 3.6 and 63.8 ± 8.2%, respectively. To further characterize whether N-type VGCC can be modulated by other calcium-binding proteins homologous to CaM, calneuron I (CalnI) was co-expressed. Our results show that CalnI and mutants without Ca2+ binding ability have an inhibitory effect on N-type VGCC, the current density was decreased from -110.1 ± 29.9 to -4.9 ± 5.0 and -3.8 ± 2.3 pA/pF, respectively. In contrast, the mutant without C terminal transmembrane domain inhibited the current to -33.1 ± 7.5 pA/pF. These results revealed the differential roles of various Ca2+ binding proteins in modulating the channel activities.